Sensory impairments and delayed regeneration of sensory axons in interleukin-6-deficient mice

Abstract

Interleukin-6 (IL-6) is a multifunctional cytokine mediating inflammatory or immune reactions. Here we investigated the possible role of IL-6 in the intact or lesioned peripheral nervous system using adult IL-6 gene knockout (IL-6(-/-)) mice. Various sensory functions were tested by applying electrophysiological, morphological, biochemical, and behavioral methods. There was a 60% reduction of the compound action potential of the sensory branch of IL-6(-/-) mice as compared with the motor branch in the intact sciatic nerve. Cross sections of L5 DRG of IL-6(-/-) mice showed a shift in the relative size distribution of the neurons. The temperature sensitivity of IL-6(-/-) mice was also significantly reduced. After crush lesion of the sciatic nerve, its functional recovery was delayed in IL-6(-/-) mice as analyzed from a behavioral footprint assay. Measurements of compound action potentials 20 d after crush lesion showed that there was a very low level of recovery of the sensory but not of the motor branch of IL-6(-/-) mice. Similar results of sensory impairments were obtained with mice showing slow Wallerian degeneration (Wlds) and a delayed lesion-induced recruitment of macrophages. However, in contrast to WldS mice, in IL-6(-/-) mice we observed the characteristic lesion-induced invasion of macrophages and the upregulation of low-affinity neurotrophin receptor p75 (p75LNTR) mRNA levels identical to those of IL-6(+/+) mice. Thus, the mechanisms leading to the common sensory deficiencies were different between IL-6(-/-) and WldS mice. Altogether, the results suggest that interleukin-6 is essential to modulate sensory functions in vivo.

Fig. 1.

Recordings of compound action potentials (CAPs) selectively measured from sensory or motor branches of the sciatic nerve. A, Schematic drawing of the recording chamber and the positions of the recording electrodes (symbolized by the differential amplifier icons) as well as stimulating electrodes (symbolized by the stippled arrow on theleft). B, CAPs recorded from sensory and motor branches of IL-6^+/+ and IL-6^−/− mice. The series of recordings was obtained by successive stimulations starting at the position 4 mm distal to the bifurcation (9/10). Between stimulations, the stimulating unit was moved at steps of 4 mm from the sciatic bifurcation in the distal direction along the nerve until reaching the final position 3–4. C, Ratio between amplitudes of CAPs derived from sensory versus motor branches.Gray column, IL-6^+/+ mice;white column, IL-6^−/− mice. The stimulating unit was positioned at compartments 8/9 as indicated inA (IL-6^+/+ mice,n = 10; IL-6^−/− mice,n = 10; p < 0.005).

Fig. 2.

Temperature sensitivities as measured by the frontpaw withdrawal time. White column, IL-6^+/+ mice; gray column, IL-6^−/− mice; black column, Wld^S mice. The time intervals until a mouse first lifted the forepaws from a plate, set at 60°C, were recorded.

Fig. 3.

Relative size distributions of L5 DRG neurons in IL-6^+/+ and IL-6^−/− mice. The size of neuronal somata was obtained from stained cross sections of the L5 DRGs. A total number of 2053 cells from IL-6^+/+(gray columns) and 1815 cells from IL-6^−/− mice (white columns) were evaluated.

Fig. 4.

Sciatic functional index (SFI) for the regeneration of crush-lesioned sciatic nerve. IL-6^+/+ mice (●); IL-6^−/−(▵) mice (error bars indicate SD).

Fig. 5.

Regeneration of motor and sensory branches 20 d after crush lesion. Series of recordings of CAPs in sensory and motor branches of IL-6^+/+, IL-6^−/−, and Wld^S mice. CAPs elicited by stimulation at the positions indicated at the left of the traces and recorded at positions 12/14 of sensory and motor branches (see Fig. 1A). The crush site was positioned between chambers 6 and 7 (bold trace).

Fig. 6.

Spatial decrement of CAPs of sensory and motor branches of regenerating sciatic nerves 20 d after crush lesion (●, IL-6^+/+ mice, n = 4; ▵, IL-6^−/− mice, n = 4; ■, Wld^S mice, n = 2). Error bars indicate SD. Amplitudes of CAPs elicited by stimulation at successive distal positions (V_dis) normalized to CAP amplitudes evoked by stimulation at position 6/7 (crush site =V_o).

Fig. 7.

Northern blot quantifications of p75LNTR mRNA levels. Samples were measured from nonlesioned and lesioned sciatic nerves of C57BL/6 (stippled column), C57BL/Wld^S (black column), IL-6^+/+ (gray column), and IL-6^−/− (white column) mice 4 d after transsection; means of three independent experiments. Error bars indicate SD. Signals were quantified by using a Phosphoimager BAS 100 (Fuji). Values were normalized to C57BL/6 as controls for Wld^S mice and to IL-6^+/+ mice as controls for IL-6^−/− mice.

Fig. 8.

Immunohistochemical staining of macrophages in sciatic nerve using F4/80 antibody. A, Cross section of nonlesioned nerve of a C57BL/6 wild-type mouse; B–D, cross sections of transsected nerve segments within 5 mm distal to the cut site, 4 d after lesion. B, Nerve segment of an IL-6^−/− mouse. C, Nerve segment of an IL-6^+/+ mouse. D, Nerve segment of a Wld^S mouse. Note the macrophage invasion (black staining, arrows) in lesioned normal and IL-6^−/− mice (black staining, arrows), which is reduced in Wld^S mice (D).